Relationship between Serum Copper, Ceruloplasmin, and Non–Ceruloplasmin-Bound Copper in Routine Clinical Practice

where n is the factor for copper bound/mg of ceruloplasmin. We retrospectively reviewed the copper, ceruloplasmin, and NCC results for 338 (150 females and 188 males) individual patients for whom both copper and ceruloplasmin testing had been requested. Nineteen patients had no liver function tests on record; of those who did, 76 were within the respective reference intervals and 243 had at least one abnormality, of which 46 were consistent with cholestasis. Serum specimens were taken in The Ipswich Hospital, where the specimens were centrifuged and the serum was refrigerated until dispatch to the Medical Toxicology Unit Laboratory, Guy’s & St Thomas’ Hospital Trust, London, United Kingdom. They subsequently analyzed the copper by flame atomic absorption on a Varian Spectra 20 (interassay CV = 5.3% at 18 μmol/L) and the ceruloplasmin by use of the Roche Tinaquant Kit on a Hitachi 912 (interassay CV = 8.9% at 300 mg/L). The reference intervals were 10–25 μmol/L and 200–500 mg/L for copper and ceruloplasmin, respectively. For each serum copper and ceruloplasmin pair, the NCC was calculated with use of a value of 0.0472 μmol/mg for n (6)(7). No patient was subsequently diagnosed by the requesting clinicians as having Wilson disease; accordingly, we were unable to calculate indices of diagnostic accuracy (with confidence intervals) such as sensitivity/specificity, likelihood ratios, and areas under ROC curves (9). The median (range) copper and ceruloplasmin results were 17 (7–41) μmol/L and 335 (180–730) mg/L, respectively (Fig. 1A1 ). The relationship between copper and ceruloplasmin was curvilinear (r2 = 0.85) but approximated linear (r2 = 0.84). The Deming linear regression equation was as follows: copper (μmol/L) = 0.052 × ceruloplasmin (mg/L) − 0.1, with the 95% confidence intervals for the slope and intercept being 0.049–0.055 and −1.1 to 0.9 μmol/L, respectively. The relationship between total copper and ceruloplasmin concentrations showed significant dispersion around the population regression line. Using the polynomial regression facility on Analyze-it™ (Analyze-it Software, Ltd), we obtained a 95% confidence interval for the serum copper concentration at a given ceruloplasmin concentration on the regression line that approximated ±4.0 μmol/L for the whole data range. More detailed examination of the data revealed that the range between the maximum and minimum serum copper values at a given ceruloplasmin concentration increased as the ceruloplasmin concentration increased. The median (range) NCC was 1.6 (−7.8 to 12.0) μmol/L. Sixty-eight NCC results were negative, and 161 results were >1.6 μmol/L (Fig. 1B1 ). The 2.5th and 97.5th percentiles for NCC were −2.9 and 6.3 μmol/L, respectively. Our data show that differences in total copper concentrations of ∼8.0 μmol/L can occur in healthy patients at a given ceruloplasmin concentration. The difference in total copper at a given ceruloplasmin concentration is large compared with the difference between the literature reference intervals for copper (∼10–12 μmol/L) (6)(7). Ceruloplasmin is a single polypeptide with 3 glucosamine-linked oligosaccharide side chains and has a mean molecular mass of 132 kDa (10) with significant size differences (11). Our data seem to be in agreement with the heterogeneity in the relationship between ceruloplasmin and total copper concentrations (11). Accordingly, any factor n used to describe the relationship for ceruloplasmin-bound copper in individual patients will have an associated confidence interval. This has important implications for the calculation of NCC and its use in routine clinical practice. A negative NCC is not theoretically possible because copper is also bound to albumin, transcuprein, and copper–amino acid complexes, but we did obtain negative values for 20.1% of patients. The upper reference limit is considered to be 1.6 μmol/L, but 47.6% of results were above this cutoff. No standardized reference method exists for ceruloplasmin, and immunologic methods used for ceruloplasmin cross-react with apoceruloplasmin. These facts, together with method-related differences, including bias, precision, and specificity, may have a larger effect on calculated NCC than previously thought. We believe that these findings require further investigation to confirm the effectiveness and robustness of NCC in clinical practice. Direct measurement of free copper is now possible (12) and may be a good approach to addressing the problems that we have found with the indirectly derived NCC. However, these methods need to be quickly evaluated for their robustness before routine use in clinical practice. Until more is known about the use of the indirectly derived NCC in routine clinical practice, we believe that if it is used, it should be used with extreme caution. Relationship between ceruloplasmin and copper concentrations (A) and NCC distribution plot (B) for 338 patients without Wilson disease.